In the coculture system of dorsal root ganglia and Schwann cells, myelination of the peripheral nervous system can be studied. This model provides experimental opportunities to observe and quantify peripheral myelination and to study the effects of compounds of interest on the myelin sheath.
The process of myelination is essential to enable rapid and sufficient signal transduction in the nervous system. In the peripheral nervous system, neurons and Schwann cells engage in a complex interaction to control the myelination of axons. Disturbances of this interaction and breakdown of the myelin sheath are hallmarks of inflammatory neuropathies and occur secondarily in neurodegenerative disorders. Here, we present a coculture model of dorsal root ganglion explants and Schwann cells, which develops a robust myelination of peripheral axons to investigate the process of myelination in the peripheral nervous system, study axon-Schwann cell interactions, and evaluate the potential effects of therapeutic agents on each cell type separately. Methodologically, dorsal root ganglions of embryonic rats (E13.5) were harvested, dissociated from their surrounding tissue, and cultured as whole explants for 3 days. Schwann cells were isolated from 3-week-old adult rats, and sciatic nerves were enzymatically digested. The resulting Schwann cells were purified by magnetic-activated cell sorting and cultured under neuregulin and forskolin-enriched conditions. After 3 days of dorsal root ganglion explant culture, 30,000 Schwann cells were added to one dorsal root ganglion explant in a medium containing ascorbic acid. The first signs of myelination were detected on day 10 of coculture, through scattered signals for myelin basic protein in immunocytochemical staining. From day 14 onward, myelin sheaths were formed and propagated along the axons. Myelination can be quantified by myelin basic protein staining as a ratio of the myelination area and axon area, to account for the differences in axonal density. This model provides experimental opportunities to study various aspects of peripheral myelination in vitro, which is crucial for understanding the pathology of and possible treatment opportunities for demyelination and neurodegeneration in inflammatory and neurodegenerative diseases of the peripheral nervous system.
In the peripheral nervous system (PNS), rapid information transduction is mediated by myelin-enwrapped axons. The myelination of axons is essential to enable the fast propagation of electric impulses, since the conduction velocity of the nerve fibers correlates to the axon diameter and myelin thickness1. Sensory signaling from the periphery to the central nervous system (CNS) relies on the activation of first-order sensory neurons that reside in enlargements of the dorsal root, termed dorsal root ganglia (DRG). For the formation and maintenance of myelin, continuous communication between axons and Schwann cells, which are the myelinating Glia cells in the PNS, is mandatory2.
Many diseases of the PNS disturb the transduction of information by either primary axonal or demyelinating damage, resulting in hypesthesia or dysesthesia. First-order sensory neurons have the ability to regenerate to an extent after neuronal damage, by a complex interaction between the neuron and surrounding Schwann cells3. In this case, Schwann cells can undergo cellular reprogramming to clear axonal as well as myelin debris and promote axonal regeneration, resulting in remyelination4. Understanding the mechanisms of myelination in health and disease is important, in order to find possible treatment options for demyelinating disorders of the PNS. Myelin can also be damaged by acute neurotrauma, and approaches to promote myelination to advance functional recovery after peripheral nerve injury are under investigation5.
Our knowledge of peripheral myelination has benefited largely from myelinating cocultures of Schwann cells and sensory neurons. Since the first approaches were applied6,7,8, myelination has been studied intensely with the use of different coculture systems9,10,11. Here, we provide a rapid and facile protocol for robust in vitro myelination of dorsal root ganglion axons. The protocol for Schwann cell preparation is based on the protocol by Andersen et al.12, previously published in Pitarokoili et al.13. We use Schwann cells derived from juvenile rats and embryonic DRG explant cultures for the coculture, in which myelination occurs at around day 14. The goal of the method is to provide a system to investigate the formation of myelin as a result of direct axon-Schwann cell interaction, and to study modulators of PNS myelination. In comparison to dissociated neuronal cell cultures, DRG explants are more anatomically preserved and form long axonal processes. Quantification of the myelinated axon area provides a sufficient readout for myelination in the coculture. The method is a valuable tool to screen therapeutic compounds for their potential effect on PNS myelination, and can also be utilized in addition to in vivo studies in animal models14.
All procedures were performed in accordance with the European Communities Council Directive for the care and use of laboratory animals.
1. Schwann cell culture
2. DRG explant culture
3. Coculture
Myelination in the coculture was assessed on days 10, 12, 14, 16, 18, and 20. The DRG explants and Schwann cells were stained for MBP, βIII-tubulin, and DAPI. The axonal network in the coculture was dense and did not change visibly in the time course of the observation. The first signs of myelin, in the form of small fragments, were detectable on day 10 and increased on day 12 (Figure 2). The MBP-positive areas increased over time until day 20 of culture. The myelination was quantified as a ratio of the MBP and βIII-tubulin positive areas. Myelination increased significantly on days 18 and 20 compared to day 10 (Figure 3; **p ≤ 0.01; ***p ≤ 0.001).
Figure 1: Appearance of Schwann cells and axons in the coculture. Exemplary picture of the coculture on day 3. Black arrows show thin DRG axons, and the white arrow points to a Schwann cell with an elongated and spindle-shaped morphology attached to an axon. Scale bar: 100 µm. Please click here to view a larger version of this figure.
Figure 2: Myelination in a coculture of DRG explants and Schwann cells. Staining for the neuronal marker βIII-tubulin and MBP was performed on days 10, 12, 14, 16, 18, and 20, after joining both cultures to investigate the development of myelination in the coculture. First signs of myelination were visible on days 10 and 12 of coculture. From day 14 on, the MBP signal was more pronounced, and myelin-enwrapped axons were detected. The myelination increased with the time of coculture until day 20. Scale bars: 100 µm. Please click here to view a larger version of this figure.
Figure 3: Quantification of myelination. On days 10, 12, 14, 16, 18, and 20 of coculture, myelination was assessed by staining axons with βIII-tubulin, and myelin with MBP. The percentage of myelinated axons was determined by calculation of the ratio of the MBP positive and βIII-tubulin stained areas. Significant differences were detected on days 18 and 20 in comparison to day 10 of coculture. Data are expressed as mean ± SEM; n = 3. One-way ANOVA with Kruskal-Wallis and Dunn's multiple comparison post-hoc test. Please click here to view a larger version of this figure.
Supplementary Figure 1: Stages of Schwann cell culture. Exemplary brightfield pictures of cultured Schwann cells (A) before and (B) after magnetic cell separation. Before magnetic cell separation, the culture includes Schwann cells with an elongated and spindle shape morphology, flat and spread-out appearing fibroblasts, and remnants of connective tissue. To achieve a purer culture of Schwann cells, magnetic cell separation is performed. Scale bars: 200 µm. Please click here to download this File.
Supplementary Figure 2: Myelination of DRG explants with and without additional Schwann cells. The MBP stained area was used to measure myelination in the coculture on day 14. The myelination of DRG explants was significantly increased if Schwann cells were added to the culture (unpaired t-test, **p ≤ 0.01). n = 3. Please click here to download this File.
Supplementary Figure 3: Gene expression analysis in the coculture. Relative gene expression levels of MBP, PMP22, MAG, Oct6, Egr2, and Olig1 were analyzed in coculture samples on day 22 by qPCR (A). MBP and PMP22 were the targets with the highest gene expression levels in the coculture. An exemplary picture of qPCR amplification products applied to an agarose gel demonstrates the qualitative abundance of the targets (B). The first and last lanes on the gel represent a DNA ladder (100 base pairs). n = 5. Please click here to download this File.
Here, we present a rapid and facile protocol for the generation of in vitro myelination by merging two separate cell type cultures, Schwann cells and dorsal root ganglion explants.
A critical step of the protocol is the cultivation of DRG explants, especially in the first days of culture. DRG are very fragile before a strong axonal network is built and must be handled very carefully, for example, when taken out of the incubator or during a change of medium. DRG that detach from the bottom of the well and are found swimming in the medium show unsuccessful cultivation. For Schwann cells, changing proliferation rates due to different cultivation conditions and supplements (e.g., serum in the medium) can represent a challenge for the coculture. If the culture is overgrown by an excessive number of Schwann cells, it detaches from the bottom of the well and becomes unusable. A titration of Schwann cell number in the culture is therefore recommended. During the establishment of the Schwann cell protocol, purity tests should be performed, using SOX10 or S100 immunostaining. In general, handling of the culture, including change of medium as well as washing and fixation steps, must be carried out carefully to ensure a successful outcome. It is important to select the time point of observation according to the research interest; day 14 represents the starting point for the first dense myelinated axons, and so can be selected as the time point of observation for the initiation of myelination, while later time points offer more complete myelin sheaths.
As this in vitro method of myelination only comprises DRG and Schwann cells, it does not exactly resemble the process of myelination in the organism. Other contributing factors, such as surrounding tissue, the microenvironment, immune cells, and signaling from distant structures, are not represented in this model. However, this method provides a suitable model to investigate myelination of the PNS by the separate analysis and manipulation of both cell types9,15,16. Schwann cells or DRG for the coculture can be harvested from diseased or treated animals to decipher the contribution of the specific cell type17,18. When using the late stages of this setup, it is highly recommended to include a control condition without additionally adding Schwann cells, to rule out the possible effects of DRG-derived Schwann cells. In our experience, myelination in DRG explants without Schwann cells is detected to a significantly lesser extent than in the coculture (Supplementary Figure 2).
The usage of DRG explants provides the advantage of intact structural architecture in comparison to dissociated neuronal cultures. Immunofluorescent staining of myelin basic protein (MBP) allows for quantifying the myelinated axon area, and provides a sufficient readout for myelination in the coculture. Gene expression analysis of coculture samples on day 22 revealed the presence of MBP, peripheral myelin protein (PMP22), myelin-associated glycoprotein (MAG), octamer-binding factor 6 (Oct6), ETS-related gene 2 (Erg2), and oligodendrocyte transcription factor 1 (Olig1), with the highest expression levels for MBP and PMP22 (Supplementary Figure 3). Hence, the described protocol provides a method with several target options for myelination markers in the coculture.
Potential applications of this method include basic research approaches on the process of myelination in the periphery, as well as the validation of therapeutic compounds for diseases of the PNS, including the damage of myelin.
The authors have nothing to disclose.
We thank Prof. Dr. Ralf Gold and PD Dr. Gisa Ellrichmann for their advice and support.
Anti-MBP, rabbit | Novus Biologicals, Centannial, USA | ABIN446360 | |
Anti-ßIII-tubulin, mouse | Biolegend, San Diego, USA | 657402 | |
Ascorbic acid | Sigma Aldrich GmbH, Steinheim, Germany | A4403-100MG | |
B27-supplement | Thermo Fisher Scientific, Schwerte, Germany | 17504-044 | |
Biosphere Filter Tip, 100 µL | Sarstedt, Nümbrecht, Germany | 70760212 | |
Biosphere Filter Tip, 1250 µL | Sarstedt, Nümbrecht, Germany | 701186210 | |
Biosphere Filter Tip, 20 µL | Sarstedt, Nümbrecht, Germany | 701114210 | |
Biosphere Filter Tip, 300 µL | Sarstedt, Nümbrecht, Germany | 70765210 | |
Bovine serum albumin | Carl Roth, Karlsruhe, Germany | 8076.4 | |
Cell strainer, 100 µM | BD Bioscience, Heidelberg, Germany | 352360 | |
Centrifuge 5810-R | Eppendorf AG, Hamburg, Germany | 5811000015 | |
CO2 Incubator Heracell | Heraeus Instruments, Hanau, Germany | 51017865 | |
Coverslips 12 mm | Carl Roth, Karlsruhe, Germany | P231.1 | |
Curved fine forceps | Fine Science Tools GmbH, Heidelberg, Germany | 11370-42 | |
DAPI fluoromount-G(R) | Biozol, Eching, Germany | SBA-0100-20 | |
Dispase II | Sigma Aldrich GmbH, Steinheim, Germany | 4942078001 | |
Distilled water (Water Purification System) | Millipore, Molsheim, France | ZLXS5010Y | |
DMEM/F-12, GlutaMAX | Thermo Fisher Scientific, Schwerte, Germany | 31331093 | |
DPBS (no Ca2+ and no Mg2+) | Sigma Aldrich GmbH, Steinheim, Germany | D8537-6X500ML | |
Ethanol | VWR, Radnor, USA | 1009862500 | |
FCS | Sigma Aldrich GmbH, Steinheim, Germany | F7524 | FCS must be tested for Schwann cell culture |
Fine forceps (Dumont #5) | Fine Science Tools GmbH, Heidelberg, Germany | 11252-20 | |
Forceps | Fine Science Tools GmbH, Heidelberg, Germany | 11370-40 | |
Forskolin | Sigma Aldrich GmbH, Steinheim, Germany | F6886-10MG | |
Gelatin | Sigma Aldrich GmbH, Steinheim, Germany | G1393-20ML | |
Gentamycin | Thermo Fisher Scientific, Schwerte, Germany | 5710064 | |
Goat anti-mouse IgG Alexa Fluor 488 | Thermo Fisher Scientific, Schwerte, Germany | A11036 | |
Goat anti-rabbit IgG Alexa Fluor 568 | Thermo Fisher Scientific, Schwerte, Germany | A11001 | |
HBSS (no Ca2+ and no Mg2+) | Thermo Fisher Scientific, Schwerte, Germany | 14170138 | |
HERAcell Incubator | Heraeus Instruments, Hanau, Germany | 51017865 | |
Heraguard ECO 1.2 | Thermo Fisher Scientific, Schwerte, Germany | 51029882 | |
Horse serum | Pan-Biotech, Aidenbach, Germany | P30-0712 | |
Image J Software | HIH, Bethesda, USA | ||
Laminin | Sigma Aldrich GmbH, Steinheim, Germany | L2020-1MG | |
Leibovitz´s L-15 Medium | Thermo Fisher Scientific, Schwerte, Germany | 11415064 | |
L-Glutamine 200 mM | Thermo Fisher Scientific, Schwerte, Germany | 25030024 | |
MACS Multistand | Miltenyi Biotec, Bergisch Gladbach, Germany | 130042303 | |
Microscissors | Fine Science Tools GmbH, Heidelberg, Germany | 15000-08 | |
Microscope | Motic, Wetzlar, Germany | Motic BA 400 | |
Microscope Axio observer 7 | Zeiss, Oberkochen, Germany | 491917-0001-000 | |
Microscope slide | VWR, Radnor, USA | 630-1985 | |
MiniMACS separator | Miltenyi Biotec, Bergisch Gladbach, Germany | 130091632 | |
MS columns | Miltenyi Biotec, Bergisch Gladbach, Germany | 130-042-201 | |
Neubauer counting chamber | Assistant, Erlangen, Germany | 40441 | |
Neuregulin | Peprotech, Rocky Hill, USA | 100-03 | |
Neurobasal medium | Thermo Fisher Scientific, Schwerte, Germany | 21103049 | |
NGF | Sigma Aldrich GmbH, Steinheim, Germany | N1408 | |
Normal goat serum | Biozol, Eching, Germany | S-1000 | |
Nunclon Δ multidishes, 4 well | Sigma Aldrich GmbH, Steinheim, Germany | D6789 | |
Paraformaldehyde | Acros Organics, New Jersey, USA | 10342243 | |
Penicillin/Streptomycin | Thermo Fisher Scientific, Schwerte, Germany | 15140-122 | |
Pipetboy | Eppendorf AG, Hamburg, Germany | 4430000018 | |
Pipettes | Eppendorf AG, Hamburg, Germany | 2231300004 | |
Poly-D-Lysin | Sigma Aldrich GmbH, Steinheim, Germany | P6407-5MG | |
Poly-L-Lysin | Sigma Aldrich GmbH, Steinheim, Germany | P4707-50ML | |
Reaction tubes, 15 mL | Sarstedt, Nümbrecht, Germany | 62554502 | |
Reaction tubes, 50 mL | Sarstedt, Nümbrecht, Germany | 62547254 | |
Reaction vessels, 1.5 mL | Sarstedt, Nümbrecht, Germany | 72690001 | |
Safety Cabinet S2020 1.8 | Thermo Fisher Scientific, Schwerte, Germany | 51026640 | |
Scissors | Fine Science Tools GmbH, Heidelberg, Germany | 14083-08 | |
Serological pipette, 10 mL | Sarstedt, Nümbrecht, Germany | 861254025 | |
Serological pipette, 25 mL | Sarstedt, Nümbrecht, Germany | 861685001 | |
Serological pipette, 5 mL | Sarstedt, Nümbrecht, Germany | 861253001 | |
Spatula | Fine Science Tools GmbH, Heidelberg, Germany | 10094-13 | |
Stereomicroscope Discovery.V8 | Zeiss, Oberkochen, Germany | 495015-0012-000 | |
Surgical scissors | Fine Science Tools GmbH, Heidelberg, Germany | 14007-14 | |
TC dish 100, cell + | Sarstedt, Nümbrecht, Germany | 833902300 | |
TC dish 35, cell + | Sarstedt, Nümbrecht, Germany | 833900300 | |
TC dish 60, cell + | Sarstedt, Nümbrecht, Germany | 833901300 | |
Thy-1 Microbeads (MACS Kit) | Miltenyi Biotec, Bergisch Gladbach, Germany | 130-094-523 | |
Triton X-100 | Sigma Aldrich GmbH, Steinheim, Germany | X100-500ML | |
Trypan Blue Solution 0.4% | Thermo Fisher Scientific, Schwerte, Germany | 15250061 | |
Trypsin (2.5%), no phenol red | Thermo Fisher Scientific, Schwerte, Germany | 15090-046 | |
Trypsin-EDTA (0.05%), phenol red | Thermo Fisher Scientific, Schwerte, Germany | 25300-054 | |
Type I Collagenase | Sigma Aldrich GmbH, Steinheim, Germany | C1639 | |
Water bath type 1008 | GFL, Burgwedel, Germany | 4285 |